Roman M. Link
This is a simple tutorial for the measurement of leaf area with ImageJ (or most likely Fiji, its powerful little brother) based on this great blog post which is meant for in-class use.
All ImageJ results tables can be saved either in ‘Comma Separated Value’ (.csv) or whitespace/tabstop separated format (generated when saving with a .xls extension, but actually just a plain text format). In either case, the output is optimized for US/UK locales, which means that points are used as a decimal separator. In order to process these files on German systems without compatibility issues, it is important to make sure that the system-wide decimal separator is correctly set before starting the analysis.
In German Windows 10, the option to change the decimal separator is well hidden:
Start Menu ➜ Windows-System ➜ Systemsteuerung ➜ Zeit und Region ➜ Region ➜ Formate ➜ Weitere Einstellungen ➜ Dezimaltrennzeichen
To avoid data compatibility problems, make sure the decimal separator is set to “.”. In this case, you will want the grouping symbol (Symbol für Zifferngruppierung) to be a comma instead of the point symbol used in Germany.
If you do not want to change your system settings, you can alternatively export everything in a .csv format and use Excel’s Daten ➜ Text in Spalten menu to manually set field delimitor and decimal separator.
In addition, before you start with the analysis, make sure that the options for analyzing threshold images are correctly set. Go to Process ➜ Binary ➜ Options and make sure that the box Black background is not marked to avoid problems with the particle analysis macro.
Open the calibration file with ImageJ (drag and drop onto the program or use the Open dialogue). The calibration file should contain a size reference with a known length. In this example, the calibration file (prepared for the analysis with a different software) contained three paper squares of known area and side length, but in theory any reference of known dimensions will suffice (e.g. a ruler or millimeter paper).
To calibrate, Select the Straight line tool and trace the size reference (in the given example, I followed the outer margin of the largest square in the calibration file, which I knew to be 4 x 4 cm²).
Set the scale by going to the Analyze ➜ Set Scale menu.
In the corresponding dialogue, set Known Distance to the known reference value (in this case, 4 cm), the Unit of length to cm and - very important - mark the box Global to make sure that the scale is the same across all opened documents.
Make sure that the image is in an 8bit black and white format (click on Image ➜ Type ➜ 8bit) to avoid problems with thresholding.
Set a black/white threshold
initial run:
after setting Size to 1-Infinity cm²:
Click on the outline file and choose File ➜ Save as ➜ Jpeg in the main window to save it (append the original filename to name_outlines.jpg)
Choose File ➜ Save as in the Results window to save the leaf area measurements as name_results.xls.
Your project folder should look like this now: